Blood preparations from which leukocytes have been removed have been utilized in blood transfusion in order to prevent non-hemolytic fever reactions such as fever, chill, itching etc. These reactions occur as side effects of blood transfusion, especially, in treatment of patients who frequently undergo blood transfusion, which result from allogeneic immunoreaction by leukocytes. Such blood preparations from which leukocytes have been removed have been produced by a centrifugation method utilizing the difference in specific gravity among blood components, or a recovery method of using a leukocyte-removing filter and the like. Among these, the method of using a leukocyte-removing filter has been used widely because blood preparations from which leukocytes are removed to a high degree can be obtained in a simple operation. Leukocytes adsorbed onto the filter were discarded along with the filter.
Meanwhile, bone marrow transplant therapy and peripheral blood stem cell transplant therapy have been applied to a hematopoietic malady occurring when chemotherapy was conducted for leukemia, cancer etc., wherein hematopoietic stem cells and hematopoietic precursor cells contained in bone marrow blood and peripheral blood have been transplanted in patients in order to overcome the hematopoietic obstacles. Further, it was found in recent years that hematopoietic stem cells and/or hematopoietic precursor cells are also contained in umbilical cord blood, and therapy by transplanting umbilical cord blood stem cells is also expected to be a promising method.
For preparing hematopoietic stem cells and hematopoietic precursor cells from a blood containing these blood components, cryopreserved blood cells are thawed to be transplanted into patients with hematopoietic obstacles (WO 96/17514). If cryopreserved blood is contaminated with erythrocytes, after the blood is thawed the erythrocytes are lyzed to cause side effects. Therefore, the erythrocytes should previously be removed from blood before freezing.
In addition, removal of platelets from blood has not been a problem so far, but in removing cells as a cause of complications in homotransplantation and removing cancer cells in autotransplantation in recent years, platelets cause aggregation and adhesion of these cells to reduce separation efficiency, so removal of platelets from blood is also desirable.
Heretofore, there is known a method of obtaining leukocytes from whole blood in which a leukocyte separation filter consisting of a fibrous material is contacted with a plasma, and a blood cell-floating fluid is passed therethrough to be separated into erythrocytes and other blood components, and the leukocytes captured in the leukocyte separation filter are recovered (Japanese Patent Publication No. 54131/1983). Further, Japanese Laid-Open Patent Publication No. 104,643/1996 discloses a method of recovering hematopoietic stem cell- and hematopoietic precursor cell-derived, erythrocyte- and platelet-free leukocytes from a blood, i.e., bone marrow blood or peripheral blood. The method comprises passing a cell population containing erythrocytes, hematopoietic stem cells and hematopoietic precursor cells through a filter which is capable of passing erythrocytes but capable of capturing leukocytes, causing a fluid flow in the opposite direction to the passing fluid, and recovering the captured leukocytes. In addition, Japanese Laid-Open Patent PublicationNo. 121,849/1987 discloses a method of obtaining hematopoietic stem cells, which comprises passing a cell population containing erythrocytes, hematopoietic stem cells, monocytes and granulocytes through a first capturing means which is capable of passing erythrocytes but capturing leukocytes, recovering the captured leukocytes in a recovery solution, passing the recovered leukocytes through a second capturing means which is capable of capturing monocytes and granulocytes but passing hematopoietic stem cells, and obtaining hematopoietic stem cells from said capturing means.
In these methods of recovering leukocytes captured in the filter described in said references, however, the method of passing a recovering solution in the same direction as that of the first passing solution requires raising the liquid pressure of the recovering solution in order to wash out leukocytes captured in the fibers, thus making it difficult to recover leukocytes in high yield. The method of recovering leukocytes captured by causing a fluid flow in the opposite direction to the first passing fluid permits spaces between the fibers to be disordered thus achieving a higher yield of leukocytes than the method of passing a fluid flow in the same direction as the first passing fluid, but nevertheless the captured leukocytes cannot sufficiently be recovered.
An object of the present invention is to provide an apparatus for high-yield recovery of leukocytes captured in a filter and a method for recovering leukocytes using the same.